Various scientific articles are referred to in parentheses throughout the specification, and complete citations are listed at the end of the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains.
Proteases are ubiquitous enzymes that play important roles in the control of cellular processes. In eukaryotes, proteases play key roles in orchestrating the progression of the cell cycle as well as in the decision process for activating programmed cell death. For example, it has become clear in the past 10 years that a large number of distinct but related cysteine proteases, called caspases, are involved in cell death activation in animals (Cryns and Yuan, 1998). From gene knock-out studies in mice, it is quite clear that different caspases play distinct roles in the cell death control of various tissues. In addition, although aspartate is the invariant residue at the P1 position of their target sites, animal caspases can be distinguished from each other by their preference of distinct substrate peptide sequences (Talanian et al. 1997). The ability to monitor the presence of different caspases in vivo should greatly facilitate our understanding of how this family of important protease may be controlled at the level of their enzymatic activity.
Many other proteases also recognize distinct targets for cleavage of proteins. These include cathepsin G, papain and thrombin, to name a few.
One in vivo approach for monitoring protease activity is the technology of Fluorescence Resonance Energy Transfer (FRET). In the approach described by Heim and Tsien (1995), two fluorescent proteins, Green Fluorescent Protein (GFP) and Blue Fluorescent Protein (BFP) are linked by a 25 amino acid linker with a trypsin cleavage site. FRET from BFP to GFP can be demonstrated with the fusion protein and is lost upon trypsin addition. The lost of FRET is measured as a decrease of green fluorescence with the concomitant increase in blue fluorescence. Although this technique can in theory be used to detect the presence of proteases in vivo, the approach is likely to suffer from lack of sensitivity. Thus, if the activity of a particular protease in a cell is low or transient, the lost of FRET in a small percentage of the expressed GFP-BFP fusion proteins may be difficult to detect. Furthermore, the assay for FRET requires sophisticated and expensive equipment and background fluorescence of particular biological organisms (such as plants) may limit the application of this technology.
From the foregoing discussion, it can be seen that there is a need for economical and sensitive screening strategies for measuring the activities of selected proteases in vivo or in cell-free extracts. Such strategies would advance the field in several respects, which include facilitating the discovery of novel proteases and drugs that can modulate specific protease activities in different cellular contexts.